The invention generally relates to small, conformationally-constrained peptide inhibitors of members of the VEGF family of compounds. Specifically, the invention provides a purified monomeric monocyclic peptide inhibitor and a purified dimeric bicyclic peptide inhibitor, both based on loop 1, 2 or 3 of VEFG-D as well as methods of making them. The invention also relates to pharmaceutical compositions and methods utilizing these peptide inhibitors.
The two major components of the mammalian vascular system are the endothelial and smooth muscle cells. The endothelial cells form the lining of the inner surface of all blood vessels and lymphatic vessels in the mammal. The formation of new blood vessels can occur by two different processes, vasculogenesis or angiogenesis (for review see Risau, W., Nature 386: 671–674, 1997). Vasculogenesis is characterized by in situ differentiation of endothelial cell precursors to mature endothelial cells and association of these cells to form vessels, such as occurs in the formation of the primary vascular plexus in the early embryo. In contrast, angiogenesis, the formation of blood vessels by growth and branching of pre-existing vessels, is important in later embryogenesis and is responsible for the blood vessel growth which occurs in the adult. Angiogenesis is a physiologically complex process involving proliferation of endothelial cells, degradation of extracellular matrix, branching of vessels and subsequent cell adhesion events. In the adult, angiogenesis is tightly controlled and limited under normal circumstances to the female reproductive system. However angiogenesis can be switched on in response to tissue damage. Importantly, solid tumors are able to induce angiogenesis in surrounding tissue, thus sustaining tumor growth and facilitating the formation of metastases (Folkman, J., Nature Med. 1: 27–31, 1995). The molecular mechanisms underlying the complex angiogenic processes are far from being understood.
Angiogenesis is also involved in a number of pathological conditions, where it plays a role or is involved directly in different sequelae of the disease. Some examples include neovascularization associated with various liver diseases, neovascular sequelae of diabetes, neovascular sequelae to hypertension, neovascularization in post-trauma, neovascularization due to head trauma, neovascularization in chronic liver infection (e.g. chronic hepatitis), neovascularization due to heat or cold trauma, dysfunction related to excess of hormone, creation of hemangiomas and restenosis following angioplasty. In arthritis, the pathological condition occurs because new capillaries invade the joint and destroy the cartilage. In diabetes, one pathological condition is caused by new capillaries in the retina invading the vitreous humour, causing bleeding and blindness (Folkman and Shing, J. Biol. Chem. 267:10931–10934, 1992). The role of angiogenic factors in these and other diseases has not yet been clearly established.
Because of the crucial role of angiogenesis in so many physiological and pathological processes, factors involved in the control of angiogenesis have been intensively investigated. A number of growth factors have been shown to be involved in the regulation of angiogenesis. These include the fibroblast growth factors (FGFs) the platelet-derived growth factors (PDGFs), the transforming growth factor alpha (TGFα), and the hepatocyte growth factor (HGF). See for example Folkman et al., J. Biol. Chem., 267: 10931–10934, 1992 for a review.
It has been suggested that a particular family of endothelial cell-specific growth factors, the vascular endothelial growth factors (VEGFs), and their corresponding receptors are primarily responsible for stimulation of endothelial cell growth and differentiation and for certain functions of the differentiated cells. These factors are members of the PDGF/VEGF family, and appear to act primarily via endothelial receptor tyrosine kinases (RTKs). The PDGF/VEGF family of growth factors belongs to the cystine-knot superfamily of growth factors, which also includes the neurotrophins and transforming growth factor-β.
Eight different proteins have been identified in the PDGF/VEGF family, namely two PDGFs (A and B), VEGF and five members that are closely related to VEGF. The five members closely related to VEGF are: VEGF-B, described in International Patent Application PCT/US96/02957 (WO 96/26736) and in U.S. Pat. Nos. 5,840,693 and 5,607,918 by Ludwig Institute for Cancer Research and The University of Helsinki; VEGF-C or VEGF2, described in Joukov et al., EMBO J., 15: 290–298, 1996, Lee et al., Proc. Natl. Acad. Sci. USA, 93: 1988–1992, 1996, and U.S. Pat. Nos. 5,932,540 and 5,935,540 by Human Genome Sciences, Inc; VEGF-D, described in International Patent Application No. PCT/US97/14696 (WO 98/07832), and Achen et al., Proc. Natl. Acad. Sci. USA, 95: 548–553, 1998; the placenta growth factor (PlGF), described in Maglione et al., Proc. Natl. Acad. Sci. USA, 88: 9267–9271, 1991; and VEGF3, described in International Patent Application No. PCT/US95/07283 (WO 96/39421) by Human Genome Sciences, Inc. Each VEGF family member has between 30% and 45% amino acid sequence identity with VEGF. The VEGF family members share a VEGF homology domain which contains the six cysteine residues which form the cystine-knot motif. Functional characteristics of the VEGF family include varying degrees of mitogenicity for endothelial cells, induction of vascular permeability and angiogenic and lymphangiogenic properties.
Vascular endothelial growth factor (VEGF) is a homodimeric glycoprotein that has been isolated from several sources. Alterative mRNA splicing of a single VEGF gene gives rise to five isoforms of VEGF. VEGF shows highly specific mitogenic activity for endothelial cells. VEGF has important regulatory functions in the formation of new blood vessels during embryonic vasculogenesis and in angiogenesis during adult life (Carmeliet et al., Nature, 380: 435–439, 1996; Ferrara et al., Nature, 380: 439–442, 1996; reviewed in Ferrara and Davis-Smyth, Endocrine Rev., 18: 4–25, 1997). The significance of the role played by VEGF has been demonstrated in studies showing that inactivation of a single VEGF allele results in embryonic lethality due to failed development of the vasculature (Carmeliet et al., Nature, 380: 435–439, 1996; Ferrara et al., Nature, 380: 439–442, 1996). The isolation and properties of VEGF have been reviewed; see Ferrara et al., J. Cellular Biochem., 47: 211–218, 1991 and Connolly, J. Cellular Biochem., 47: 219–223, 1991.
In addition VEGF has strong chemoattractant activity towards monocytes, can induce the plasminogen activator and the plasminogen activator inhibitor in endothelial cells, and can also induce microvascular permeability. Because of the latter activity, it is sometimes referred to as vascular permeability factor (VPF). VEGF is also chemotactic for certain hematopoetic cells. Recent literature indicates that VEGF blocks maturation of dendritic cells and thereby reduces the effectiveness of the immune response to tumors (many tumors secrete VEGF) (Gabrilovich et al., Blood 92: 4150–4166, 1998; Gabrilovich et al., Clinical Cancer Research 5: 2963–2970, 1999).
Vascular endothelial growth factor B (VEGF-B) is very strongly expressed in heart and only weakly in lung, whereas the reverse is the case for VEGF. Reverse transcriptase-polymerase chain reaction (RT-PCR) assays have demonstrated the presence of VEGF-B mRNA in melanoma, normal skin, and muscle. This suggests that VEGF and VEGF-B, despite the fact that they are co-expressed in many tissues, have functional differences. Gene targeting studies have shown that VEGF-B deficiency results in mild cardiac phenotype, and impaired coronary vasculature (Bellomo et al., Circ. Res. 86:E29–35, 2000).
Human VEGF-B was isolated using a yeast co-hybrid interaction trap screening technique by screening for cellular proteins which might interact with cellular retinoic acid-binding protein type I (CRABP-I). The isolation and characteristics including nucleotide and amino acid sequences for both the human and mouse VEGF-B are described in detail in PCT/US96/02957, and in U.S. Pat. Nos. 5,840,693 and 5,607,918 by Ludwig Institute for Cancer Research and The University of Helsinki and in Olofsson et al., Proc. Natl. Acad. Sci. USA, 93: 2576–2581, 1996.
VEGF-C was isolated from conditioned media of the PC-3 prostate adenocarcinoma cell line (CRL1435) by screening for ability of the medium to produce tyrosine phosphorylation of the endothelial cell-specific receptor tyrosine kinase VEGFR-3 (Flt4), using cells transfected to express VEGFR-3. VEGF-C was purified using affinity chromatography with recombinant VEGFR-3, and was cloned from a PC-3 cDNA library. Its isolation and characteristics are described in detail in Joukov et al., EMBO J., 15: 290–298, 1996.
VEGF-D was isolated from a human breast cDNA library, commercially available from Clontech, by screening with an expressed sequence tag obtained from a human cDNA library designated “Soares Breast 3NbHBst” as a hybridization probe (Achen et al., Proc. Natl. Acad. Sci. USA, 95: 548–553, 1938). Its isolation and characteristics are described in detail in International Patent Application No. PCT/US97/14696 (WO98/07832)
In PCT/US97/14696, the isolation of a biologically active fragment of VEGF-D, designated VEGF-DΔNΔC, is also described. This fragment consists of VEGF-D amino acid residues 93 to 201 linked to the affinity tag peptide FLAG®. The entire disclosure of the International Patent Application PCT/US97/14696 (WO 98/07832) is incorporated herein by reference.
The VEGF-D gene is broadly expressed in the adult human, but is certainly not ubiquitously expressed. VEGF-D is strongly expressed in heart, lung and skeletal muscle. Intermediate levels of VEGF-D are expressed in spleen, ovary, small intestine and colon, and a lower expression occurs in kidney, pancreas, thymus, prostate and testis. No VEGF-D mRNA was detected in RNA from brain, placenta, liver or peripheral blood leukocytes.
PlGF was isolated from a term placenta cDNA library. Its isolation and characteristics are described in detail in Maglione et al., Proc. Natl. Acad. Sci. USA, 88: 9267–9271, 1991. Presently its biological function is not well understood.
VEGF3 was isolated from a cDNA library derived from colon tissue. VEGF3 is stated to have about 36% identity and 66% similarity to VEGF. The method of isolation of the gene encoding VEGF3 is unclear and no characterization of the biological activity is disclosed.
Similarity between two proteins is determined by comparing the amino acid sequence and conserved amino acid substitutions of one of the proteins to the sequence of the second protein, whereas identity is determined without including the conserved amino acid substitutions.
A major function of the lymphatic system is to provide fluid return from tissues and to transport many extravascular substances back to the blood. In addition, during the process of maturation, lymphocytes leave the blood, migrate through lymphoid organs and other tissues, enter the lymphatic vessels, and return to the blood through the thoracic duct. Specialized venules, high endothelial venules (HEVs), bind lymphocytes again and cause their extravasation into tissues. The lymphatic vessels, and especially the lymph nodes, thus play an important role in immunology and in the development of metastasis of different tumors. Unlike blood vessels, the embryonic origin of the lymphatic system is not clear, and at least three different theories exist as to its origin. Lymphatic vessels are difficult to identify due to the absence of known specific markers available for them.
Lymphatic vessels are most commonly studied with the aid of lymphography. In lymphography, X-ray contrast medium is injected directly into a lymphatic vessel. The contrast medium gets distributed along the efferent drainage vessels of the lymphatic system and is collected in the lymph nodes. The contrast medium can stay for up to half a year in the lymph nodes, during which time X-ray analyses allow the follow-up of lymph node size and architecture. This diagnostic technique is especially important in cancer patients with metastases in the lymph nodes and in lymphatic malignancies, such as lymphoma. However, improved materials and methods for imaging lymphatic tissues are needed in the art.
As noted above, the PDGF/VEGF family members act primarily by binding to receptor tyrosine kinases. In general, receptor tyrosine kinases are glycoproteins which comprise an extracellular domain capable of binding a specific growth factor(s), a transmembrane domain, which is usually an alpha-helical portion of the protein, a juxtamembrane domain, which is where the receptor may be regulated by, e.g., protein phosphorylation, a tyrosine kinase domain, which is the enzymatic component of the receptor, and a carboxy-terminal tail, which in many receptors is involved in recognition and binding of the substrates for the tyrosine kinase.
Five endothelial cell-specific receptor tyrosine kinases have been identified, belonging to two distinct subclasses: three vascular endothelial cell growth factor receptors, VEGFR-1 (Flt-1), VEGFR-2 (KDR/Flk-1), VEGFR-3 (Flt4), and the two receptors of the Tie family, Tie and Tie-2 (Tek). These receptors differ in their specificity and affinity. All of these have the intrinsic tyrosine kinase activity which is necessary for signal transduction.
The only receptor tyrosine kinases known to bind VEGFs are VEGFR-1, VEGFR-2 and VEGFR-3. VEGFR-1 and VEGFR-2 bind VEGF with high affinity, and VEGFR-1 also binds VEGF-B and PlGF. VEGF-C has been shown to be the ligand for VEGFR-3, and it also activates VEGFR-2 (Joukov et al., The EMBO Journal, 15: 290–298, 1996) . VEGF-D binds to both VEGFR-2 and VEGFR-3. A ligand for Tek/Tie-2 has been described in International Patent Application No. PCT/US95/12935 (WO 96/11269) by Regeneron Pharmaceuticals, Inc. The ligand for Tie has not yet been identified.
Recently, a novel 130–135 kDa VEGF isoform specific receptor has been purified and cloned (Soker et al., Cell, 92: 735–745, 1998). The VEGF receptor was found to specifically bind the VEGF165 isoform via the exon 7 encoded sequence, which shows weak affinity for heparin (Soker et al., Cell, 92: 735-745, 1998). surprisingly, the receptor was shown to be identical to human neuropilin-1 (NP-1), a receptor involved in early stage neuromorphogenesis. PlGF-2 also appears to interact with NP-1 (Migdal et al., J. Biol. Chem., 273: 22272–22278, 1998).
VEGFR-1, VEGFR-2 and VEGFR-3 are expressed differently by endothelial cells. Generally, both VEGFR-1 and VEGFR-2 are expressed in blood vessel endothelia (Oelrichs et al., Oncogene, 8: 11–18, 1992; Kaipainen et al., J. Exp. Med., 178: 2077–2088, 1993; Dumont et al., Dev. Dyn., 203: 80–92, 1995; Fong et al., Dev. Dyn., 207: 1–10, 1996), and VEGFR-3 is mostly expressed in the lymphatic endothelium of adult tissues (Kaipainen et al., Proc. Natl. Acad. Sci. USA, 9: 3566–3570, 1995). VEGFR-3 is also expressed in the blood vasculature surrounding tumors.
Although VEGFR-1 is mainly expressed in endothelial cells during development, it can also be found in hematopoetic precursor cells during early stages of embryogenesis (Fong et al., Nature, 376: 66–70, 1995). In adults, monocytes and macrophages also express this receptor (Barleon et al., Blood, 87: 3336–3343, 1995). In embryos, VEGFR-1 is expressed by most, if not all, vessels (Breier et al., Dev. Dyn., 204: 228–239, 1995; Fong et al., Dev. Dyn., 207: 1–10, 1996).
The receptor VEGFR-3 is widely expressed on endothelial cells during early embryonic development, but as embryogenesis proceeds becomes restricted to venous endothelium and then to the lymphatic endothelium (Kaipainen et al., Cancer Res., 54: 6571–6577, 1994; Kaipainen et al., Proc. Natl. Acad. Sci. USA, 92: 3566–3570, 1995). VEGFR-3 is expressed on lymphatic endothelial cells in adult tissues. This receptor is essential for vascular development during embryogenesis.
The essential, specific role in vasculogenesis, angiogenesis and/or lymphangiogenesis of VEGFR-1, VEGFR-2, VEGFR-3, Tie and Tek/Tie-2 has been demonstrated by targeted mutations inactivating these receptors in mouse embryos. Disruption of the VEGFR genes results in aberrant development of the vasculature leading to embryonic lethality around midgestation.
Analysis of embryos carrying a completely inactivated VEGFR-1 gene suggests that this receptor is required for functional organization of the endothelium (Fong et al., Nature, 376: 66–70, 1995). However, deletion of the intracellular tyrosine kinase domain of VEGFR-1 generates viable mice with a normal vasculature (Hiratsuka et al., Proc. Natl. Acad. Sci. USA, 95: 9349–9354, 1998). The reasons underlying these differences remain to be explained but suggest that receptor signalling via the tyrosine kinase is not required for the proper function of VEGFR-1.
Analysis of homozygous mice with inactivated alleles of VEGFR-2 suggests that this receptor is required for endothelial cell proliferation, hematopoesis and vasculogenesis (Shalaby et al., Nature, 376: 62–66, 1995; Shalaby et al., Cell, 89: 981–990, 1997).
Targeted inactivation of both copies of the VEGFR-3 gene in mice resulted in defective blood vessel formation characterized by abnormally organized large vessels with defective lumens, leading to fluid accumulation in the pericardial cavity and cardiovascular failure at post-coital day 9.5 (Dumont et al., Science, 282: 946–949, 1998). On the basis of these findings, it has been proposed that VEGFR-3 is required for the maturation of primary vascular networks into larger blood vessels. However, the role of VEGFR-3 in the development of the lymphatic vasculature could not be studied in these mice because the embryos died before the lymphatic system emerged. Nevertheless it is assumed that VEGFR-3 plays a role in development of the lymphatic vasculature and lymphangiogenesis given its specific expression in lymphatic endothelial cells during embryogenesis and adult life. This is supported by the finding that ectopic expression of VEGF-C, a ligand for VEGFR-3, in the skin of transgenic mice, resulted in lymphatic endothelial cell proliferation and vessel enlargement in the dermis. Furthermore this suggests that VEGF-C may have a primary function in lymphatic endothelium, and a secondary function in angiogenesis and permeability regulation which is shared with VEGF (Joukov et al., EMBO J., 15: 290–298, 1996).
In addition, VEGF-like proteins have been identified which are encoded by four different strains of the orf virus. This is the first virus reported to encode a VEGF-like protein. The first two strains are NZ2 and NZ7, and are described in Lyttle et al., J. Virol., 68: 84–92, 1994. A third is D1701 and is described in Meyer et al., EMBO J., 18: 363–374, 1999. The fourth strain is NZ10 and is described in International Patent Application PCT/US99/25869. It was shown that these viral VEGF-like proteins bind to VEGFR-2 on the endothelium of the host (sheep/goat/human) (Meyer et al., EMBO J., 18: 363–374, 1999; and Ogawa et al. J. Biol. Chem., 273: 31273–31282, 1988) and this binding is important for development of infection (International Patent Application PCT/US99/25869). The entire disclosure of the International Patent Application PCT/US99/25869 is incorporated herein by reference. These proteins show amino acid sequence similarity to VEGF and to each other.
The orf virus is a type of species of the parapoxvirus genus which causes a highly contagious pustular dermatitis in sheep and goats and is readily transmittable to humans. The pustular dermatitis induced by orf virus infection is characterized by dilation of blood vessels, swelling of the local area and marked proliferation of endothelial cells lining the blood vessels. These features are seen in all species infected by orf and can result in the formation of a tumor-like growth or nodule due to viral replication in epidermal cells. Generally orf virus infections resolve in a few weeks, but severe infections that fail to resolve without surgical intervention are seen in immune impaired individuals.
The biological functions of the different members of the VEGF family are currently being elucidated. Of particular interest are the properties of VEGF-D and VEGF-C. These proteins bind to both VEGFR-2 and VEGFR-3—localized on vascular and lymphatic endothelial cells respectively—and are closely related in primary structure (48% amino acid identity) Both factors are mitogenic for endothelial cells in vitro.
VEGF-C has been shown to be angiogenic in the mouse cornea model and in the avian chorioallantoic membrane (Cao et al., Proc. Natl. Acad. Sci. USA 95: 14389–14394, 1998) and was able to induce angiogenesis in the setting of tissue ischemia (Witzenbichler et al., Am. J. Pathol. 153: 381–394, 1998). Furthermore, VEGF-C stimulated lymphangiogenesis in the avian chorioallantoic membrane (Oh et al. , Dev. Biol. 188: 96–109, 1997) and in a transgenic mouse model (Jeltsch et al., Science 276: 1423–1425, 1997).
VEGF-D was shown to be angiogenic in the rabbit cornea (Marconcini et al., Proc. Natl. Acad. Sci. USA 96: 9671–9676, 1999) The lymphangiogenic capacity of VEGF-D has not yet been reported, however, given that VEGF-D, like VEGF-C, binds and activates VEGFR-3, a receptor thought to signal for lymphangiogenesis (Taipale et al. , Curr. Topics Micro. Immunol. 237: 85–96, 1999), it is highly likely that VEGF-D is lymphangiogenic.
VEGF-D and VEGF-C may be of particular importance for the malignancy of tumors, as metastases can spread via either blood vessels or lymphatic vessels; therefore molecules which stimulate angiogenesis or lymphangiogenesis could contribute toward malignancy. This has already been shown to be the case for VEGF. It is noteworthy that VEGF-D gene expression is induced by c-Fos, a transcription factor known to be important for malignancy (Orlandini et al., Proc. Natl. Acad. Sci. USA 93: 11675–11680, 1996). It has been speculated that the mechanism by which c-Fos contributes to malignancy is the up-regulation of genes encoding angiogenic factors.
Each monomer of the VEGF dimer resembles other cystine-knot proteins, having an elongated structure consisting of pairs of twisted, anti-parallel β-strands connected by a series of solvent-exposed loops. The crystal structure of the complex between VEGF and the immunoglobulin-like domain 2 of VEGFR-1 (Wiesmann et al., Cell 91: 695–704, 1997) and mutational analyses (Muller et al., Proc. Natl. Acad. Sci. USA 94: 7192–7197, 1997; Keyt et al., J. Biol. Chem. 271: 5638–5646, 1996) of VEGF indicate that residues important for binding of this molecule to its receptors VEGFR-1 and VEGFR-2 are located primarily on the solvent-exposed loops at the ends of each monomer. The VEGF monomers associate to form disulfide-linked dimers in a side-by-side, head-to-tail fashion, thus creating two symmetrical clusters of receptor binding residues, one at each “pole” of the VEGF dimer.
There is great interest in the development of pharmacological agents which antagonize the receptor-mediated actions of VEGFs (Martiny-Baron and Marme, Curr. Opin. Biotechnol. 6: 675–680, 1995). Monoclonal antibodies to VEGF have been shown to suppress tumor growth in vivo by inhibiting tumor-associated angiogenesis (Kim et al., Nature 362: 841–844, 1993). Similar inhibitory effects on tumor growth have been observed in model systems resulting from expression of either antisense RNA for VEGF (Saleh et al., Cancer Res. 56: 393–401, 1996) or a dominant-negative VEGFR-2 mutant (Millauer et al., Nature 367: 576–579, 1994). While indicating the potential of interfering with the VEGF signalling system as a chemotherapeutic approach, these approaches—being protein- and DNA-based—are not optimal given current pharmaceutical delivery technologies. The development of small molecule antagonists of members of the VEGF family would offer distinct advantages over protein- and DNA-based strategies in terms of oral absorption, penetration across cell membranes, bioavailabilty and biological half life.